PROJECT SUMMARY How the brain performs its computational task is a great unsolved problem in biology, but this answer is vital for us to understand and combat disorders of brain function like autism, schizophrenia, and Alzheimer?s. One appealing strategy towards solving this problem is to deconstruct the brain into the component parts?the cell types?and to determine their respective key features and dissect which physiological functions are sub served by each distinct type. Recently, advances in single cell RNA-sequencing technologies have catalyzed the identification of dozens of transcriptomically distinct cell types in the mammalian neocortex, many of which share homology between human and mouse, and it will likely soon be possible to have a complete transcriptomic taxonomy of neocortical cell types for multiple mammalian species. However, the ability to probe the function of most of these refined cell types is lacking, especially in human. As a result, the defining functional characteristics of human neocortical cell types remain largely unknown. In order to address this shortcoming, the current proposal lays out a comprehensive strategy to generate a first-in-class toolbox of cell type-specific genetic tools. Using recently developed methods to explore chromatin landscapes as well as Allen Institute-generated single-cell transcriptomics datasets, Aim 1 seeks to identify conserved neocortical cell type- and cell class-specific cis-regulatory modules across adult mouse and human. Candidate cis-regulatory modules near cell type- and cell class-specific marker genes identified in Aim 1 will be filtered for sequence conservation and accessibility conservation. In Aim 2, top candidates from Aim 1 will be used to generate cell class-specific viral vector libraries and screened for reporter expression in adult mouse neocortex, followed by secondary verification of individual on-target reporter vectors in adult mouse and human neocortex. In Aim 3 the most promising subset of these tools will be more deeply evaluated and validated using single cell RNA-seq and patch clamp electrophysiology on labeled cell populations in parallel for adult mouse and human neocortex. On the whole this project will generate a toolbox of novel reagents for the interrogation of neocortical cell type function that is compatible with diverse mammalian species, including mouse, monkey, and human. As such, these tools will not only enable direct cross-species functional comparisons of presumed orthologous neocortical cell types, but may also be suitable for human gene therapy applications. If successful, these tools will be of exceptional value and a welcome new resource for the neuroscience community.